POLYTENE AND DIPLOID INTERPHASE CHROMOSOMES

拟议的研究建立在最近扩展我们的硬件和软件，允许，在一个广义的分辨率重叠的方式，在光和电子显微镜水平的亚细胞结构的三维重建。我们应用这种方法来分析高阶染色体的结构和功能，使用独特的间期多线染色体的果蝇。 我们专注于细胞学鉴定的多线染色体带，interbands和泡芙（转录活性，形态独特的染色体位点）。 我们利用果蝇变态，昆虫激素蜕皮激素触发，作为一种方式来针对一个特征的转录级联。新技术，三维条形码，提供了一个系统的方法来研究细胞核中的三维二倍体间期染色体地形。 在活细胞核中，染色体动力学现在可以用绿色荧光蛋白（GFP）标记特定的染色体位点来研究。 本提案的具体目标是：（A）继续用电子显微断层摄影术测定特定多线染色体带、带间带和蓬松物的三维结构。 B.）研究脉冲标记的DNA复制位点的体内结构动力学（定量运动分析），随后是蜕皮激素诱导的粉扑形成和消退。 C.）的应用三维条形码技术测定果蝇早期胚胎细胞核中二倍体染色体的三维构型。 D.）在眼睛成虫盘中逐细胞基础上分析PEV遗传扰动内的二倍体染色体核地形图。 E.）研究4维动力学的二倍体染色体行为使用新可用的GFP染色体位点标记方法。

The proposed research builds on recent extensions to our hardware and software that allows, in a generalized resolution overlapping fashion, 3-dimensional reconstructions of subcellular structures at both the light and electron microscopic levels. We apply this methodology to the analysis of higher-order chromosome structure and function using the unique interphase polytene chromosomes of Drosophilia melanogaster. We focus on cytologically identified polytene chromosome bands, interbands and puffs (transcriptionally active, morphologically distinctive chromosome sites). We utilize Drosophila metamorphosis, triggered by the insect hormone ecdysone, as a way to target a characteristic transcriptional cascade. New technology, a 3-dimensional Bar Code, provides for a systematic approach to the study of 3-dimension diploid interphase chromosome topography in the nucleus on a cell-by-cell basis. Chromosome dynamics, in living nuclei, can now be studied using green fluorescent protein (GFP) to label specific chromosomal loci. The specific aims of this proposal are: A.) Continue the determination of the 3-dimensional structure of specific polytene chromosome band, interbands and puffs using electron microscope tomography. B.) Study the in vivo structural dynamics (quantitative motion analysis) of pulse labeled DNA replication sites followed by ecdysone induced puff formation and regression. C.) Determine the 3-dimensional diploid chromosome topography in Drosophila early embryo nuclei using the 3- dimensional Bar Code methodology. D.) Analyze diploid chromosome nuclear topography within a PEV genetic perturbation on a cell- by-cell basis in eye imaginal disks. E.) Study the 4-dimensional dynamics of diploid chromosome behavior using the newly available GFP chromosome locus labeling methodology.